Oral Mucosal Electroporation Device and Use Thereof

ABSTRACT

The present invention relates to electroporation (EP) devices that are able to generate an electroporation causing electrical field at the mucosal layer, and preferably in a tolerable manner. Further, it includes the generation of a protective immune response, cellular and/or humoral, using the oral EP device along with a genetic construct that encodes an immunogenic sequence.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of application Ser. No. 13/641,977filed Oct. 18, 2012, which is a 371 National stage entry ofInternational Application No. PCT/US2011/034277, filed Apr. 28, 2011,and claims benefit of U.S. Provisional Application No. 61/328,868, filedApr. 28, 2010, the contents of which are incorporated herein byreference.

FIELD OF INVENTION

The present invention relates to electroporation devices that enable thedelivery of therapeutics to a subject.

BACKGROUND

A vast majority of human pathogens are known to initiate infections atmucosal surfaces, thus, making the gastrointestinal, urogenital andrespiratory tracts major routes of entry into the body. As a result, theother primary way to contract an infection is through blood-borne routessuch injections, transfusions and bites. Examples of mucosally-infectingagents include cold viruses, influenza, food poisoning agentstuberculosis, sexually transmitted diseases, cholera, diphtheria and theplague.

The mucous membranes are one of the largest organs of the body.Collectively, they cover a surface area of more than 400 m² (equivalentto one and half tennis courts) and comprise the linings of thegastrointestinal, urogenital and respiratory tracts. These mucosalsurfaces, while located inside the body, are actually a physical barrierbetween the outside and the sterile interior cavity of the body known asthe “systemic” environment. Critical nutrients, oxygen and othermolecules are constantly taken up across these mucosal barriers;however, another important function of the mucous is to keep invadingpathogens out. Daily these mucous membranes are bombarded by outsideelements and it is up to the unique immune system of the mucous todetermine what is potentially harmful and what is beneficial.

The importance of mucosal immunology lies in the interplay between themucosal response and the systemic immune response. Several studies havedemonstrated that stimulating the immune system systemically (i.e. viainjection or blood-borne routes) results in the production of protectiveantibody and T cells only within the sterile, internal environment ofthe body-no mucosal response is generated. On the other hand,stimulation of the mucosal immune response can result in production ofprotective B and T cells in both mucosal and systemic environments sothat infections are stopped before they get into the body.

The mucous membranes produce a special type of antibody called secretoryIgA or sIgA. The mucous membranes are bathed in huge quantities of sIgA,which act as a first line of defense to neutralize invading pathogens.Experimental evidence shows that the presence of sIgA correlates withresistance to infection by various pathogens, including bacteria,viruses, parasites and fungi. It has also been shown to neutralizeviruses and prevent their adherence to the epithelial cells lining themucous (thereby preventing infection) as well as mediating excretion ofpathogens and preventing the assembly of mature virus particles.

Another important component of mucosal immunity is the T cell-mediatedimmune response. T-cells that specifically recognize pathogens can helpantibodies to clear the infection or directly kill the invaderthemselves. T cells produced in the mucous are capable of travelingthroughout the mucosal tissues through special “homing” receptors ontheir membranes. This means that if an immune response is generated inthe gastrointestinal lining, T cells produced there can travel to othermucosal sites, for example, the lungs or nasal cavity, providingprotection over a large area.

Despite the important role of the mucosal surface, only a handful ofvaccines specifically target this area of the immune system, thus thereremains a need for vaccines that are directed toward the mucosal surfaceto provide protective immune responses at the mucosal tissue.

SUMMARY OF THE INVENTION

There are provided electroporation devices capable of electroporatingcells of a mucosal membrane of a mammal. Such devices include anelectrode microneedle plate, a counter electrode plate, a main housingand an energy source. The main housing is in physical communication withsaid microneedle plate and counter electrode plate, wherein the mainhouse is in fluid communication with a syringe capable of storing apharmaceutical formulation for delivery. The energy source is inelectrical communication with the microneedle plate and capable ofgenerating an electric potential and delivering the electric potentialto the cells through the microneedle plate.

In another aspect, there are provided methods of administering apharmaceutical formulation to cells of a mucosal membrane of a mammalwith the provided devices. The methods comprise contacting saidmicroneedle plate to said mucosal membrane, delivering saidpharmaceutical formation to said mucosal membrane, and applying anelectroporation causing electrical pulse to the mucosal membrane throughthe microneedle plate, which was generated by said energy source.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an immunization (via standard injection) and challengetimeline to be performed in a mouse.

FIG. 2 displays a graph that shows that chemokine adjuvants inducecellular immunity specific against influenza APR/8/34 in a mouse modelof mucosal lung infection.

FIG. 3a displays a graph that shows InfluenzaA/PR/8/34-specific serumlong-lived IgA and IgG pre-challenge; FIG. 3b displays a graph thatshows InfluenzaA/PR/8/34 neutralizing antibody pre-challenge; FIG. 3cdisplays a line graph that shows average weight loss over days; and FIG.3d displays a line graph that shows the various survival rates afterchallenge.

FIG. 4 displays a timeline and additional information about IndianRhesus Macaques Immunization Schedule.

FIG. 5 displays a graph that shows ELISpot data from known IM(intramuscular)/EP (electroporation) delivery of DNA vaccine is superiorto IM delivery alone.

FIGS. 6a-6d display graphs and images that show a strong immune responsewas generated: FIG. 6a displays a graph that shows ELISpot data; FIG. 6bdisplays a graph that shows levels of Tcell proliferation; FIG. 6c showsplates from R10 or SIV peptide cultures; and FIG. 6d shows graphs thatsuggest CFSE Proliferation.

FIGS. 7a and 7b display graphs that show that CTACK Co-immunizationAugments Cytokine Secretion by CD4+ T cells in the BAL: FIG. 7a displaysgraphs that show the cellular response in the periphery; FIG. 7bdisplays graphs that show the cellular response in BAL.

FIGS. 8a-8d display graphs that show that CTACK Elicits High Levels ofCytokine Secreting CD8+ T cells in the Lung: FIG. 8a displays a graphthat shows the 107a+CD8+ levels; FIG. 8b displays a graph that shows theIFN-gamma CD+ levels; FIG. 8c displays a graph that shows the TNF+CD8+levels; and FIG. 8d displays a graph that shows IL-2+CD8+ levels.

FIG. 9 displays a photo that shows positive GFP expression by way offluorescence.

FIG. 10 displays a 4×4 array (Inovio Pharmaceuticals, Inc., Blue Bell,Pa.)

FIGS. 11a and 11b display graphs that show HAI titer levels in serumfrom macaques that were immunized with SynCon™ influenza vaccine.Results shown are two weeks post-second immunization: FIG. 11a shows HAItiters with respect to A/H1N1/Mexico/2009 strain; and FIG. 11b shows HAItiters with respect to A/H1N1/New Caledonia.

FIG. 12a displays photos that show GFP expression in guinea pig oralmucosal tissue following shallow injection of GFP plasmid andelectroporation Whole cheek mounts were harvested 3 days post-treatmentand viewed under a fluorescent microscope to determine positive GFPexpression.

FIG. 12b displays a graph that shows HS-specific IgA titers following 3immunizations in the guinea pig.

FIG. 13 is a ¾ view of an oral electroporation/injection devicecomprising: a pulse generator, an injection and an electroporationdevice.

FIG. 14 is a drawing showing a vertical cross-section A-A of the oralelectroporation/injection device.

FIG. 15 is an exploded assembly of the electroporation/injection device.

FIG. 16 is the main electrode micro-needle plate of theelectroporation/injection device.

FIG. 17 shows the OM-I/EP device in relation to an open mouth.

FIG. 18a displays a graph showing IgA titers in Saliva; FIG. 18bdisplays a graph showing IgA titers in Stool; and FIG. 18c displays agraph showing IgA titers in Blood.

DETAILED DESCRIPTION OF THE INVENTION

There are provided electroporation devices capable of electroporatingcells of a mucosal membrane of a mammal. Such devices include anelectrode microneedle plate, a counter electrode plate, a main housingand an energy source. The main housing is in physical communication withsaid microneedle plate and counter electrode plate (item #4), whereinthe main house is in fluid communication with a syringe capable ofstoring a pharmaceutical formulation for delivery. The energy source(item #10) is in electrical communication with the microneedle plate andcapable of generating an electric potential and delivering the electricpotential to the cells through the microneedle plate. In an embodiment,there is also a piston in physical communication between said mainhousing and said microneedle plate. The piston is actuatable and byactuating can cause even distribution of the pharmaceutical formulationthrough the microneedle plate.

In one aspect of the invention, there are provided oral electroporation(EP) devices that are able to generate an electroporation causingelectrical field at the mucosal layer, and preferably in a tolerablemanner. In one embodiment of this aspect, there is an oral mucosalinjection and electroporation device (OM-I/EP) that is adapted toperform delivery of therapeutic (or prophylactic) formulations, such asDNA vaccines, and the transfection into the mucosal tissue/cells on theinside of the mouth. During a DNA vaccination procedure the device wouldbe affixed across the cheek area of the patient. The main body with themain electrode micro-needle plate feature on the inside of the mouth andthe return electrode plate clamp feature adjacent, on the outside of thecheek. The DNA vaccine would be injected through the micro-needle plate;this would then be followed by low voltage EP pulses applied to thatsame electrode micro-needle plate, this design co-locates the DNAvaccine and the electroporation to the same area. Research has shownthat the co-location of DNA vaccine and EP to be very important in theamount of DNA vaccine transfection into the surrounding cells.

In some embodiments, the microneedles of the microneedle plate are madefrom electrically conductive materials comprising gold and silver platedbrass, gold and silver plated copper, stainless steel, or titanium, orother commonly known conductive metal or metal-like material. In someembodiments, the energy source is capable of delivering through themicroneedle plate to the cells of the mucosal membrane at least onepulse of electrical energy having characteristics of between 1V and 30V,2 mA and 100 mA, or 1 mS and 250 mS. The mucosal membrane or mucosaltissue can be buccal, nasal, esophageal, rectal, vaginal, vulva,intestinal, bowel, stomach, bladder, urinary tract, or eye tissue, andpreferably buccal tissue, e.g., the inner surface of the mouth.

In another aspect, there are provided methods of administering apharmaceutical formulation to cells of a mucosal membrane of a mammalwith the provided devices. The methods comprise contacting saidmicroneedle plate to said mucosal membrane, delivering saidpharmaceutical formation to said mucosal membrane, and applying anelectroporation causing electrical pulse to the mucosal membrane throughthe microneedle plate, which was generated by said energy source.

During in vivo electroporation, electric pulses are applied directly tothe tissue to enhance uptake of extracellular molecules. Present typesof in vivo EP are done with very high volt/centimeter electrical fieldstrengths, using such large electrical field strengths is would bepainful to the patient in mucosal tissue due to the high density ofnerves. With the current OM-l/EP devices, they can be equipped todeliver very low field strength EP, such as using the low energyelectrical pulses that were applied at intradermal (ID) injection sites,which were described in an earlier filed, co-owned PCT applicationentitled, “CONTACTLESS ELECTROPERMEABILIZATION ELECTRODE AND METHOD”having application number PCT/US10/31431, filed Apr. 16, 2010, andincorporated by reference herein in its entirety. Such intradermal EPcan be performed with very low voltages and with minimal to no pain tothe patient. In early experiments on mucosal tissues these lower EPfield strengths have shown transfection into mucosal tissue with similarresults (data not shown). The EP parameters can include voltages rangingfrom 0.1 volts (V) to 30 V, 0.1 V to 20 V, 0.1 V to 15 V, 0.1 V to 10 V,0.1 V to 9 V, 0.1 V to 8 V, 0.1 V to 7 V, 0.1 V to 6 V, 0.1 V to 5V, 0.1V to 4V, 0.1 V to 3V, 0.1 V to 2V, 0.1 V to 1 V, 2V to 30V, 2 V to 20 V,2V to 15 V, 2V to 10V, 2 V to 9 V, 2 V to 8 V, 2 V to 7 V, 2 V to 6 V,2V to 5V, 2 V to 4V, 2V to 3V, 4V to 30V, 4V to 20V, 4V to 15V, 4V to10V, 4V to 9V, 4 V to 8V, 4V to 7V, 4V to 6V, 4V to 5V, 6V to 30V, 6V to20V, 6V to 15V, 6V to 10V, 6V to 9V, 6V to 8V, 8V to 30V, 8V to 20V, 8Vto 15V, 8V to 10V, 8V to 9 V, 10 V to 30 V, 10 V to 20 V, or 10 V to 15V; and currents ranging from 2 mA to 100 mA, 3 mA to 100 mA, 4 mA to 100mA, 5 mA to 100 mA, 6 mA to 100 mA, 7 mA to 100 mA, 8 mA to 100 mA, 9 mAto 100 mA, 10 mA to 100 mA, 20 mA to 100 mA, 30 mA to 100 mA, 40 mA to100 mA, 60 mA to 100 mA, 80 mA to 100 mA, 2 mA to 80 mA, 3 mA to 80 mA,4 mA to 80 mA, 5 mA to 80 mA, 6 mA to 80 mA, 7 mA to 80 mA, 8 mA to 80mA, 9 mA to 80 mA, 10 mA to 80 mA, 20 mA to 80 mA, 30 mA to 80 mA, 40 mAto 80 mA, 60 mA to 80 mA, 2 mA to 60 mA, 3 mA to 60 mA, 4 mA to 60 mA, 5mA to 60 mA, 6 mA to 60 mA, 7 mA to 60 mA, 8 mA to 60 mA, 9 mA to 60 mA,10 mA to 60 mA, 20 mA to 60 mA, 30 mA to 60 mA, 40 mA to 60 mA, 2 mA to40 mA, 3 mA to 40 mA, 4 mA to 40 mA, 5 mA to 40 mA, 6 mA to 40 mA, 7 mAto 40 mA, 8 mA to 40 mA, 9 mA to 40 mA, 10 mA to 40 mA, 20 mA to 40 mA,30 mA to 40 mA, 2 mA to 30 mA, 3 mA to 30 mA, 4 mA to 30 mA, 5 mA to 30mA, 6 mA to 30 mA, 7 mA to 30 mA, 8 mA to 30 mA, 9 mA to 30 mA, 10 mA to30 mA, 20 mA to 30 mA, 2 mA to 20 mA, 3 mA to 20 mA, 4 mA to 20 mA, 5 mAto 20 mA, 6 mA to 20 mA, 7 mA to 20 mA, 8 mA to 20 mA, 9 mA to 20 mA, 10mA to 20 mA, 2 mA to 10 mA, 3 mA to 10 mA, 4 mA to 10 mA, 5 mA to 10 mA,6 mA to 10 mA, 7 mA to 10 mA, 8 mA to 10 mA, 9 mA to 10 mA, 2 mA to 9mA, 3 mA to 9 mA, 4 mA to 9 mA, 5 mA to 9 mA, 6 mA to 9 mA, 7 mA to 9mA, 8 mA to 9 mA, 2 mA to 8 mA, 3 mA to 8 mA, 4 mA to 8 mA, 5 mA to 8mA, 6 mA to 8 mA, 7 mA to 8 mA, 2 mA to 7 mA, 3 mA to 7 mA, 4 mA to 7mA, 5 mA to 7 mA, 6 mA to 7 mA, 2 mA to 6 mA, 3 mA to 6 mA, 4 mA to 6mA, 5 mA to 6 mA, 2 mA to 5 mA, 3 mA to 5 mA, 4 mA to 5 mA, 2 mA to 4mA, or 3 mA to 4 mA. In some embodiments the EP parameters used rangefrom 30 volts and 100 mA on the high end to 2 volts and 2 mA on the lowend. For EP delivery, the desired tissue received two (2) pulses 100 mseach with a 100 ms delay between pulses.

The OM-I/EP device has a main electrode micro needle plate (item #3 &FIG. 15) fastened to the head of a main housing (item #1). A voltagereturn electrode plate (item #4) and arm (items #5 and #6) is placedadjacent and outside the mouth. The main housing (item #1) can bemounted to a standard 1-ml lure-lock syringe (item #8). In use, themicro needle plate array (item #3 & FIG. 15) is placed on the inside ofthe mouth in intimate contact with the buccal mucosal lining (innersurface of cheek). The voltage return electrode (item #4) would be incontact with the outside adjacent surface of the cheek. The micro needleplate array (item #3 & FIG. 15), main housing (item #1) and the attachedsyringe (item #8) form the injection/electroporation device. The designof the large micro needle array allows for the injection of DNA vaccineover a large area. The small size and short length of the micro needlesplaces the DNA vaccine to a controlled specific depth. A piston (item#2) and its sealing a-ring (item #9) form a common manifold area anddriver that will insure even distribution of DNA vaccine through themicro-needle plate (item #3 & FIG. 15).

The requirement for the main electrode (Item #3 & FIG. 15) is that theyhave many micro-needles of a specific length and diameter. The electrodemust also, be made from electrically conductive materials (such asgold/silver plated brass or copper, stainless steel and/or titanium).The DNA vaccine must be placed in the upper most layers of the mucosamembranes. The main electrode (item #3 & FIG. 15) can be made by a fewmanufacturing techniques: such as Chemical etching, Electrical Dischargemachining (EDM) and Electro-less nickel plating on a sacrificialpattern. The main housing and support parts could be made from injectionmolded materials (such as ABS, Polycarbonate and Polyolefin).

FIGS. 4-8 d show results that support the following:

Optimized SIV DNA constructs+EP elicited IFN-g (˜12,000 SFC/106) andproliferative CD8+ T cell responses (˜20%) (no difference with CTACK).These responses were highest following the 4th immunization. Theaddition of optimized CTACK DNA did not further enhance the inducedresponse in the periphery by:

-   -   IFN-g ELISpot    -   CFSE Proliferation    -   PBMC cytokine secretion    -   IgA in the sera

The addition of optimized CTACK DNA changes the phenotype of theresponse in the mucosa as measured by:

-   -   BAL cytokine secretion    -   More Polyfunctional CD8+ T cells    -   Higher Frequencies of responding CD4+ and CD8+ T Cells    -   IgA in Fecal & BAL samples

Examples

The present invention is further illustrated in the following Examples.It should be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, various modifications of the invention in addition tothose shown and described herein will be apparent to those skilled inthe art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Experiments were performed to assess IgA titers in the blood, nasalsecretions, saliva and stools of animals immunized via an EP enhancedmucosal (orally) route with Influenza HA antigens. Significant IgAtiters observed in the saliva is indicative of a mucosal immune responsebeing successfully raised in a local mucosal region. Detection of IgAresponses in the stool samples indicates a mucosal response at a distantsite was raised. Detection of IgA titers in the blood sera suggests asystemic response was also raised.

H5 IgA ELISA

Following three mucosal EP-enhanced immunizations, positive H5 specificIgA titers were observed in the saliva of 3 out of 4 animal'selectroporated with the 4×4 device (Inovio Pharmaceuticals, Inc., BlueBell, Pa.) and 4 out of 4 animals electroporated with a caliperelectroporation device. One animal was positive in the injection onlygroup. See FIGS. 18a -18 c.

Two animals had target specific positive IgA titers in their bloodsamples following three immunizations with the 4×4 device.

One animal from both the 4×4 device and caliper groups had targetspecific IgA responses in their stools.

None of the negative controls or injection only group animals hadpositive IgA stool or blood samples.

1.-8. (canceled)
 9. Method of electroporating cells of a mucosalmembrane of a mammal, wherein the device comprises: contacting themucosal membrane with a plurality of microneedles of an electrodemicroneedle plate; positioning a voltage return electrode in contactwith an opposite side of the mucosal membrane that is across from theelectrode microneedle plate; delivering a pharmaceutical solutionthrough the electrode microneedle plate and into the mucosal membrane;generating an electric potential at an energy source, wherein theelectric potential is sufficient to electroporate the cells; anddelivering at least one pulse of electrical energy having the electricpotential from the energy source, through the electrode microneedleplate, through the mucosal membrane, and through the voltage returnelectrode.